Hemoglobin switching is an example of tissue specific and temporal regulation of gene expression. In humans, embryonic globin chains (zeta and epsilon) are expressed during the first trimester of gestation followed by a switch to the expression of fetal globin chains early in the second trimester and adult globin chains after birth. Previous work performed in the Laboratory of Chemical Biology has identified a silencer 200 to 400 bp upstream of the epsilon-globin gene which has been implicated in suppressing epsilon globin gene expression during the fetal and adult stages. Our work has focused on characterizing proteins binding to the silencer and the function of the binding of trans-acting factors to the silencer. We are in the process of elucidating the mechanism(s) that trans-acting factors interacting with the silencer utilize in order to regulate epsilon-globin transcription. We are also studying the interactions of the silencer with other regulatory elements controlling epsilon-globin gene expression. Using DNase I footprinting, we have identified several sites of protein binding to the silencer. The major protected region shares a high percentage of homology with the binding sites of an erythroid specific transcription factor GATA-1 and a yeast silencer binding protein, ABF-1. Using gel mobility shift assays with K562 nuclear extract, yeast ABF-1 protein and a probe bracketing the major protected region and competitor DNA with mutations in the sites homologous to GATA-1 and ABF-1 binding sites we have demonstrated that a protein, likely GATA-1, binds to the GATA site in the silencer and that another protein, likely a human homologue of the yeast ABF-1 protein, binds to the silencer. We are currently in the process of examining the function of mutations made in the yeast ABF-1 and GATA binding sites. We are also in the process of cloning the human homolog of the yeast ABF-1 gene.